Packing system and method for chromatography columns

ABSTRACT

A method for providing an aseptic chromatography column includes the steps of pre-sterilizing an empty chromatography column, pre-sterilizing a chromatography medium, introducing the pre-sterilized chromatography medium into the pre-sterilized chromatography column using aseptic equipment, thus providing an aseptic chromatography column comprising chromatography medium.

FIELD OF THE INVENTION

The present invention relates to a system for packing chromatographycolumns with a chromatography medium and packing method for use in suchcolumns. More specifically, the invention relates to packing systems andmethods for improving the packing of chromatography media intochromatography columns.

BACKGROUND OF THE INVENTION

Columns used in liquid chromatography typically comprise a tubular bodyenclosing a porous chromatography medium through which a carrier liquidflows, with separation taking place by material collection between thecarrier liquid and solid phase of the porous medium. Prior to anyseparation process, the bed has to be prepared starting from the slurryof particles that has to be introduced into the column. The process ofbed formation is called ‘the packing procedure’ and a correctly packedbed is a critical factor influencing the performance of a columncontaining a packed bed. The goal of the packing procedure is to providea bed compressed by the optimum amount of compression—the optimumcompression factor.

In detail, the porous medium is formed by consolidating a suspension ofdiscrete particles, known as “slurry” that is pumped or poured or suckedinto the column, usually from one end. Consolidation of the slurry intoa packed bed is typically achieved by filtering it against a particleretaining filter and further compressing the formed filter cake so thatit is packed into a volume which is less than the volume that it wouldhave occupied if it had sedimented under the influence of only gravityto form a sedimented bed. The degree of compression depends upon thetype of chromatography medium and typically ranges from in between2-20%. The efficiency of subsequent chromatographic separations relieson the liquid distribution and collection system at the fluid inlet andoutlet of the packed bed, but primarily on the homogeneity and stabilityof the packed bed formed. If the packed bed is not homogeneous andstable a deleterious effect will be experienced for chromatographicseparations performed on the bed. Homogeneity and stability of thepacked bed depend upon the optimum degree of compression, which must bedetermined experimentally for each column size (width or diameter), bedheight and bed medium.

Several methods are known in the art for packing columns (see, forexample, US 2003/0146159). “Flow packing” is a method typically utilisedfor the preparation of analytical columns (i.e. columns of about 1-10 mmcolumn diameter) and semi-preparative columns (i.e. columns of 10-100 mmcolumn diameter) or even larger. In flow packing, one end of a column isclosed by a frit or a filter. At the other end, a slurry or suspensionof the packing material is pumped or poured into the column tube. Afiltration bed builds up against the frit and grows until a filter cakehas formed. The bed is then compressed further to its “target bedheight” by percolating a number of column volumes (ca. 3-10) of apacking solvent at a flow rate that is higher than the flow rates usedin operation. Consolidation and subsequent compression take place underthe influence of the seepage force, that is the reaction of the bed tothe pressure gradient required to maintain the flow rate of the streamof liquid percolating through the bed. Once the bed is compressed by theflow, the flow is stopped, the outlet at the bottom of the column isclosed and an adapter or upper end cell is adjusted to the target heightof the compressed bed. This adjustment is done quickly to avoid arelaxation of the compressed bed exceeding the target bed height.

The flow packing method has the disadvantage that beds of packingmaterial compressed in this manner are axially heterogeneous during theflow compression step yielding highest compression close to the outletof the column and zero compression at the top of the packed bed. Thisresults in a major relaxation of the bed and a possible re-arrangementof the particles once the packing flow has been stopped and the upperend cell has been brought into position. The gradient in bed compressioninherent to this method may result in poor bed stability and poor columnefficiency depending on the type of medium and the packed bed geometry.

Standard methods of flow packing may not be suitable for the wide borecolumns used in preparative chromatography. Among other factors, it isoften undesirable to design equipment that requires application of apacking flow rate and thus a packing pressure substantially higher thanthe pressure required for subsequent operation. To remedy this problem,packing methods relying on mechanical axial compression are used. Axialcompression methods achieve the bed compression by an axial movement ofthe adapter (end cell). Hereby, the need for high liquid pressure in thecolumn space during packing is removed. A further advantage of the axialcompression method is that the bed is compressed homogeneously in axialdirection, which avoids the problems of relaxation and particlere-arrangements that occur with the flow packing method.

Radial gradients in bed compression and bed voidage occur with bothmethods described, which is due to wall friction effects. The impact ofthe radial heterogeneity depends on the bed geometry, i.e. the ratio ofdiameter to height. As described above, it is the gradient ofcompression and bed voidage in axial direction that is substantiallydifferent between the flow packing method and the axial compressionmethod.

A disadvantage of axial compression is that columns packed using thismethod require a means for moving the end cell and a means forcontrolling this movement. Typical methods for the movement are motordrives or hydraulic systems. As these are attached to or built in to thecolumn, the cost and mechanical complexity of axial compression columnsis substantially higher than for flow packing columns.

Prior to any consolidation and compression the medium has to beintroduced into the column. Large scale columns are preferably preparedby injecting slurry through a central slurry nozzle or valve into thecolumn. This enables a closed system approach which is preferable forsanitary reasons. Columns based on a slurry valve design may be designedfor axial compression packing using a movable adapter or for flowpacking using a fixed end cell.

DEFINITIONS

“Analyte” shall be defined as a substance, compound or chemical ofnatural or synthetic origin, or a reaction product or derivative ormetabolite thereof. For the avoidance of doubt, the term shall includebiological molecules, such as proteins, peptides, amino acids andnucleic acids, and synthetic molecules such as drugs and/or pro-drugs.

“Medium” shall be defined as any material in which a chromatographicseparation is effected. Examples of medium include, but are not limitedto, materials which effect ion exchange chromatographic separation, sizeexclusion chromatographic separation, affinity binding chromatographicseparation and reverse phase chromatographic separation.

A “disposable” column is characterized by a pre-treatment of thechromatography medium in order to reduce installation andqualification/validation work otherwise required with non-disposablecolumns. As a minimum, the pre-treatment involves the formation of thebed of porous medium. Additional pre-treatment can be reduction ofmicrobiological burden, sterilization, depyrogenation etc.

Disposable column may be used as single-use columns, which means thatthe user is not performing cleaning regimes that require qualification(e.g. testing, validation, etc) of the packed bed before repeated use.

One embodiment of a disposable column is a complete column that isdelivered pre-packed with chromatography medium.

Another embodiment of a disposable column consists of a first devicerepresenting a frame or vessel designed to resist pressure and loadexerted on one or multiple lateral surfaces of the packed bed duringoperation in order to provide dimensional stability for a packed bed,and a second device, representing a container, shell, cartridge, bag orthe like containing the porous medium or bed that is attached to thefirst device for operation. With the latter embodiment, the porousmedium is contained in the secondary container and can be replaced whilethe frame is re-usable. In this case, the degree of compression of theporous medium required for operation may be adjusted after inserting thecontained medium into the frame (see, for example US2002/0166816 andWO2005/009585).

By “non-integral to and external to the column drive means” is meant adrive means which is a separate entity which is only used when packingthe column and is removed or detached from the column in its operation.Thus, when the end user operates the column the drive means is notpresent and attached to the column.

SUMMARY OF THE INVENTION

The object of the invention is to provide a packing method whichovercomes the drawbacks of the prior art methods.

The main advantage of the invention is that the packed bed is preparedby the axial compression method yielding advantages of improved bedstability and high column efficiency without the need for highmechanical complexity in the column construction required byconventional solutions for axial compression columns. This allows for asubstantial cost reduction.

Yet another advantage of the method of the invention is that it iscompatible with low-pressure fluid handling equipment (e.g. aperistaltic pump) as the compression is achieved primarily by theexternal drive means, such as a compression frame, rather than by thehigh pressures required by flow compression.

Yet another advantage of the method of the invention is that thepacking/filling of the column can be performed as a closed system, whichmeans that the column may even be filled/packed under aseptic conditionsstarting from a pre-sterilized column and pre-sterilized chromatographymedium. The method can therefore be used on “ready-to-process” or“disposable” columns.

Still a further advantage is that the chromatography column is scalable(i.e. increasing or decreasing the column size leads to a predictableperformance). This is due to the fact that the filling of the column viathe slurry nozzle is done prior to the axial compression step at anelevated adapter position. This allows for homogeneous distribution ofthe slurry over the cross-sectional area of the column compared to flowpacking using a nozzle based on one designed with fixed end cells (forexample, see U.S. Pat. No. 6,524,484). For the latter, the packing hasto be performed at a constant bed height, the target bed height, whichmay lead to radial gradients in packed bed density and compression,respectively.

Another advantage of the method of the invention is that the column doesnot need to be as long as axial compression columns of the prior artbecause not all of the slurry needs be introduced into the column priorto the commencement of bed formation.

A further advantage of the invention is that a minimum adapter (endunit) stroke is required compared to systems using known methods andthus allows for a more compact construction.

According to a first aspect of the invention, there is provided a methodfor providing an aseptic chromatography column, said method comprisingthe steps of:

-   -   pre-sterilize an empty chromatography column;    -   pre-sterilize a chromatography medium;    -   introducing the pre-sterilized chromatography medium into the        pre-sterilized chromatography column using aseptic equipment,        thereby providing an aseptic chromatography column comprising        chromatography medium.

According to a second aspect of the invention, there is provided anaseptic chromatography column comprising chromatography medium whereinthe chromatography column has been pre-sterilized separately and thechromatography medium has been pre-sterilized separately beforeintroduction into the chromatography column.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic transverse sectional view of a chromatographycolumn of the prior art showing the basic features thereof.

FIG. 2 is a three dimensional schematic showing a transverse sectionalview of a chromatography column which can be packed using a methodaccording to the invention.

FIG. 3 shows a three dimensional schematic showing a transversesectional view of a different embodiment of a chromatographic columnwhich can be packed using a method in accordance with the invention.

FIG. 4 is a three-dimensional schematic of a chromatography columnpacked using a method in accordance with the invention.

FIG. 5 is a schematic illustration of a system for packing an axial flowchromatography column using a method in accordance with the invention.

FIG. 6 is a chromatogram showing the chromatographic separation ofacetone on a chromatography column packed in accordance with theinvention, both in upflow (dotted line) and downflow (solid line) mode.

FIG. 7 describes a method for calculating the reduced plate height andasymmetry factor from an eluted peak.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows schematically the general components of a chromatographycolumn 1 as known from the prior art (for example, see U.S. Pat. No.6,524,484). The column has a cylindrical fluid-impermeable side wall 11,e.g. of stainless steel or a high-strength/reinforced polymeric materialwhich may be translucent. The open top and bottom ends of the side wall11 are closed by top and bottom end assemblies or units 12, 13. Each endunit has a fluid-impermeable end plate 3 fitting sealingly to plug theopening of the cylindrical wall 11, and preferably made of stainlesssteel or high-strength engineering plastics material, e.g.polypropylene. The end plates are backed up by retaining metal plates 2bearing against their outer surfaces and projecting radially beyond theside wall as retaining flanges 22 through which adjustable tension rods14 are secured. These link the top and end assemblies 12, 13 and helpthe construction to withstand high fluid pressures.

Each end plate 3 has a central through-opening 31 for communicationbetween the exterior of the column and the packing bed space 9 definedby the side wall 11 and end assemblies 12, 13. Access through theopening 31 is subdivided into separate conduits, connected externallythrough a connection manifold 8.

A filter layer 4, typically of filtered or woven plastics or steel,extends across the area of the bed space 9 at the inner surface of theend plate 3. The inner surface 35 of the end plate 3 is recessed behindthe filter layer 4, e.g. conically as illustrated, and preferably withthe use of support ribs (not indicated) supporting the filter layer 4from behind, to define between them a distribution channel 34. One ofthe communication conduits, a mobile phase conduit 33, opens inwardlyinto this distribution channel 34, as well as outwardly to a mobilephase connector 81 of the manifold 8.

From the manifold 8, an access valve device 5 projects inwardly throughthe end plate opening 31 and sealingly through a central orifice 41 ofthe filter layer 4. The access valve 5, governs the communication of oneor more conduits from the manifold 8 directly to the bed space 9, i.e.bypassing the filter layer 4. Indicated here are first and second valvedconduits 51, 61 governed by the valve 5, and connected externallythrough connectors 82 of the manifold 8.

A packed bed of particulate stationary phase material fills the bedspace 9 between the top and bottom filter layers 4. The packed bed maybe formed by the “flow packing” method described above while thestationary phase is introduced into the bed space in the form of aslurry via access valve 5, preferably at the upper end piece of the ofthe column. The excess liquid passes through the bed, filter layer 4,distribution channel 34, conduit 33 and is removed through connector 81(arrow B). As the stationary phase is retained by filter layer 4, thebed is growing throughout the process. Consolidation and subsequentcompression take place under the influence of the seepage force (i.e.the reaction of the bed to the pressure gradient required to maintainthe flow rate of the stream of liquid percolating through the bed). Thegrowing bed is compressed substantially by introducing the slurry at avery high flow rate. By optimizing the packing flow rate, the desiredcompression factor can be achieved for the packed bed. Once the bed iscompressed by the flow and the desired amount of stationary phase hasbeen added to the column, the slurry flow is stopped and the accessvalve 5 is closed. This method does not require any adjustment of theupper end plate 3 such that the end plate is at a fixed bed height(target bed height) throughout the process.

An alternative method for compressing the bed of stationary phase is toapply mechanical axial compression by an axial movement of the adapteror end plate 3. This is achieved by means of a motor drive or hydraulicsystem attached to or built into the column (not shown in FIG. 1).

After column packing the valve devices 5 being closed, a mobile phase isfed in through mobile phase connector 81 (arrow “A”), passes throughconduit 33 into the distribution channel 34 and through the filter layer4 to elute down through the packed bed, effecting separation of itscomponents or analytes. Liquid eluate passes thought the filter layer 4of the bottom end assembly 13 and out through the mobile phase connector81 thereof (arrow “B”) for collection as appropriate. While this is anexample of “downflow” chromatography, in that chromatographic separationis effected by the downward movement of the mobile phase through thecolumn, the skilled person will understand that separation mayalternatively be achieved by “upflow” chromatography, simply by pumpingmobile phase upwards through the column and thus reversing the directionof flow. In this mode, mobile phase would enter the column at connector81 (arrow “B”), move upwards through the stationary phase or particulatemedium, and be collected from connector 81 (arrow “A”) at the top of thecolumn.

FIG. 1 and the above explanation are to illustrate general relationshipsof components and a typical mode of operation. The skilled person willunderstand, and it will also appear from the following description, thatother specific constructions and modes of operation may be appropriatefor different kinds of process.

FIG. 2 shows a transverse sectional view of a column in accordance withthe invention. The column 101 comprises a tubular housing 111, a firstend unit 112 (partially shown) and a second end unit 113, securedtogether to form a fluid tight seal by means of o-rings 107/108 andtension rods 114 having heads 116. First filter 104 and second filter106 are adjacent to the first end unit 112 and second end unit 113,respectively. These filters 104, 106, together with side wall 111,define a bed space 109 for containing a bed of particulate medium.

The housing 111 and end units 112, 113 are typically composed ofstainless steel or a high-strength plastic material such aspolypropylene. In a preferred embodiment, where the column is to be usedfor the separation of biologically active substances, the material isbiologically inert such that it does not elicit an immune response inhumans in accordance with United States Pharmacopia (USP)<88> class VI.Tension rods 114, with heads 116, secure the end units 112, 113 to thehousing 111 to form a fluid-tight bed space 109 which is capable ofwithstanding high operating pressures.

Filters 104, 106 are each positioned on the interior face of the endunits 112, 113 and act to define the bed space (together with side wall111) and to prevent leakage of particulate medium from the bed space109. The end units 112 and 113 (and hence the first 104 and second 106filters which are adjacent thereto) are axially movable in relation toeach other. In FIG. 2, end unit 112 is axially movable within thehousing 111 relative to the second end unit 113 but it will beunderstood that other embodiments are possible where one or both of theend units (and hence filters 104, 106) are axially movable relative tothe other.

The bed space 109 is packed with a bed of particulate medium by firstadjusting the height of the first end unit 112 such that the distancebetween the first 104 and second 106 filters is greater than the targetbed height. In this state, the tie rods 114 and heads 116 are notattached to the column. The distance between the end units is secured bymeans of an external drive or compression frame (not shown). A slurry orsuspension of particulate medium is then introduced into the column viavalve means 120, the valve means comprising a central bore 121 andnozzle 124. At the same time as the suspension is being added to the bedspace 109, excess liquid may also be removed from the bed space 109 viaport 140 to produce a settled bed of particulate medium. Afterintroduction of the desired amount of stationary phase, valve means 120and nozzle 124 are closed. The settled bed is then compressed by theaxial movement of the end unit 112 and filter 104 to achieve the targetbed height, effected by an external drive or compression frame (notshown). The external drive, which is not an integral part or the columnconstruction, may be controlled manually or by software means in orderto achieve the desired target bed height. The end units are then securedagainst each other by help of the tie rods (114) and heads (116),respectively, and the column is then released from the external drive orcompression frame.

In FIG. 2 the nozzle 124 is shown in its filling position being exposedto the bed space 109 but it will be understood that it can be retractedto a closed position within the top end plate 112 after filling of thecolumn. The nozzle 124 and valve means 120 are locked in either open orclosed position by a locking device (not shown). A wide range of nozzlescan be used which facilitate the distribution and even packing of slurrywithin the bed space. One alternative for achieving an open/closedfunctionality at the packing valve and nozzle respectively is to have anozzle that is fixed in the bed space (and thus not retractable) andlocated adjacent to a moveable element or sleeve on the inside oroutside of the nozzle that opens and/or closes the nozzle depending uponits position.

Mobile phase or liquid containing one or more analytes or substances forseparation on the column is added via first port 133. The liquid thenpasses through the first filter 104 into the bed space 109 that has beenpacked with particulate medium as described above. Chromatographicseparation of analyte(s) which has been introduced onto the particulatemedium in this manner is effected by introduction of, and elution by,mobile phase. The mobile phase will finally exit the column throughsecond filter 106 and via second port 140. The resulting fractions ofmobile phase, which contain different analytes, can then be collected.

It will be understood by the skilled person that the column may beoperated in either a “downflow” mode, as described above, or in an“upflow” mode where the direction of flow of the mobile phase isreversed such that it moves up the column. In upflow mode, mobile phasewill enter the column via second port 140, move upwards through the bedof particulate medium in bed space 109, to exit the column forcollection at first port 133.

FIG. 3 is a transverse sectional view of a column in accordance with theinvention. The column is similar to the column of FIG. 2, with many ofthe features being identical to those described in that figure. Thus thecolumn has a valve means 220 for the addition of a suspension ofparticulate medium into a bed space 209 and a first port 233 for theaddition or collection of mobile phase. However, the column differs fromthat embodiment previously described in that it has a second port 240comprising a passageway 242 which extends through second end unit 213to, and is in fluid communication with (via hollow member 260), bedspace 209 from which liquid can be added or collected. As is evidentfrom the figure, the second port 240 is at essentially the same level orelevation as the first port 233, thus facilitating the addition andcollection of mobile phase to/from the column. This arrangement hasfurther advantages in that it assists in the installation of the column,decreases the risk of siphoning and reduces the likelihood ofintroduction of air into the column.

The arrangement of the component parts are otherwise as described inFIG. 2. The column 201 comprises a tubular housing 211, a first end unit212 (partially shown) and a second end unit 213, secured together toform a fluid tight seal by means of o-rings 207/208 and tension rods 214with heads 216. First filter 204 and second filter 206 are adjacent tothe first end unit 212 and second end unit 213, respectively. Thesefilters 204, 206, together with side wall 211, define a bed space 209for containing a bed of particulate medium. The first end unit 212 andthe second end unit 213 are axially movable with respect to each otherby means of an external drive (not shown) such as an actuator, press orframe which may be operated manually or automatically under the controlof software. The filters 204, 206 are adjacent to the first end unit 212and second end unit 213 and are axially movable with the end unitsrelative to each other. In the embodiment shown, only end unit 212 andfirst filter 204 are axially movable relative to the second end unit 213and filter 206 but it will be understood that other embodiments arepossible wherein either or both of the end units and filters are axiallymovable relative to the other.

The column is packed with a bed of particulate medium to a target bedheight as described in FIG. 2. The height of the first end unit 212 isadjusted such that the distance between the first 204 and second 206filters is greater than the target bed height by means of an externaldrive (not shown). A slurry or suspension of particulate medium is thenintroduced into the column via valve means 220, the valve meanscomprising a central bore 221 and nozzle 224. Excess liquid may beremoved from the bed space 209 via passageway 242 and port 240 toproduce a settled bed of particulate medium. The settled bed is thencompressed by the axial movement of the end unit 212 and filter 204,effected by the external drive (not shown), to achieve the target bedheight. The external drive may be controlled manually or by softwaremeans in order to achieve the desired target bed height. The end unitsare then secured against each other by help of the tie rods (214) andheads (216), respectively.

Mobile phase or liquid containing one or more analytes or substances forseparation on the column is added via first port 233. The liquid thenpasses through the filter 204 into the bed space 209 that is packed withparticulate medium as describe above. Chromatographic separation ofanalyte(s) which has been introduced onto the particulate medium in thismanner is effected by introduction of, and elution by, mobile phase. Themobile phase will finally exit the column through second filter 206 andvia passageway 242 to second port 240. The resulting fractions of mobilephase, which contain different analytes, can then be collected.

It will be understood by the skilled person that the column may beoperated in either a “downflow” mode, as described above, or in an“upflow” mode where the direction of flow of the mobile phase isreversed such that it moves up the column. In upflow mode, mobile phasewill enter the column via second port 240, move along passageway 242 andupwards through the bed of particulate medium in bed space 209, to exitthe column for collection at first port 233.

In the embodiment shown, hollow member 260 is an integral part of thecolumn. However, it will be understood that by means of connectors andappropriate tubing made from a suitable material (e.g. polypropylene,polyurethane, etc.) that the hollow member 260 need not be integral tothe column.

The application and collection of mobile phase at the same elevation ona single end unit simplifies use, in terms of operator access andhandling, reduces the risk of air accessing the system and decreases thespace necessary to set up the column.

FIG. 4 presents a three dimensional schematic representation of columnof FIG. 3 of larger column diameter, from which the external features ofthe column are evident. The column comprises a first end unit 317, whichis movable during the compression step, second end unit 318 and housing311 which are secured together to form a fluid-tight seal by tensionrods 314 and heads 316. Particulate medium in the form of a slurry canbe introduced into the bed space (not shown) via valve means 320. Firstport 333 serves as a conduit for mobile phase or liquid containinganalyte(s) to be separated on the particulate medium. Hollow member 360,which is in fluid communication with the bed space via an outlet at thebase of the column (not shown), ends in second port 340 from whichappropriate fractions of mobile phase eluted from the column may becollected. As can be seen, second port 340 is at essentially the samelevel or elevation as the first port 333 through which mobile phase canbe introduced (or collected). This arrangement facilitates useroperation and sample handling. In the embodiment shown in FIG. 4, thecapacity of the column is approximately 10 liters; it will be understoodthat a wide range of column capacities is possible, typically rangingfrom 0.1 to 2000 liters. Preferred capacities when using the column as adisposable column are in the range of 0.5 to 50 liters.

FIG. 5 is a schematic illustration of a system for packing an axial flowchromatography column using a method in accordance with the invention.The system comprises a column 401 as described in FIG. 2 above. Thecolumn is connected to an external drive means 470 (such as acompression frame) which, by means of platforms 472 and 474 can adjustthe height of an axially movable first end unit (not shown) and firstfilter (404) within the bed space 409 of the column relative to a secondend unit (not shown) and second filter 406.

The system is initially primed with liquid from container 480 via pump485 which is in fluid communication with outlet valve 450 to facilitatethe removal of air from the column or from at least the bottomdistribution system and filter layer. Excess liquid or air may beremoved from the column via the upper inlet/outlet port of the column401 to exit as waste 460. Pressure sensor 490 monitors pressure withinthe column during the priming phase and/or subsequent packing phase.

The first end unit and filter 404 are initially adjusted to positionH_(o) (shown as 474 _(O) in the figure) such that the distance betweenthe first and second filters 404, 406 is greater than that of the targetbed height within the bed space 409. Particulate medium, in the form ofa slurry or suspension within container 430, is then introduced into thecolumn bed space 409 under pressure by pump 435 through inlet valve 420.Any residual slurry remaining within container 430 can be washed intothe bed space 409 by rinsing the container 430 with a packing liquidfrom reservoir 431 via valve 433. At the same time as slurry is beingintroduced into the bed space 409, excess liquid may be removed viaoutlet 440 in order to produce a settled bed of medium within the bedspace. The excess liquid passes through outlet valve 450 and thence isvoided as waste 460. During the consolidation step the pressure in thecolumn bed space 409 is monitored by sensor 490 and can be regulated byadjusting the flow rate of pump 435 in order to achieve differentdegrees of pre-compression of the packed bed.

Finally, compression of the bed to its target bed height is achieved bylowering the platform 474 to position H_(F) (shown as 474 _(F) in thefigure) which causes axial movement of the first filter 404 relative tothe second filter 406. Depending on the type of chromatography medium,the packing process may be controlled to different degrees ofpre-compression by flow packing and final compression by axialcompression. After lowering the upper end unit to the final positionH_(F), the column end units are secured by means of the tie rods and thecolumn can be released from the compression frame.

FIG. 5 and the above explanation are to illustrate the method of packingthe columns. The skilled person will understand that other arrangementsof pumps, vessels and sensors are possible. The use of disposable liquidhandling equipment, like bags and tubing, is preferred when packingdisposable columns that shall fulfill requirements on a controlled levelof microbiological burden or columns that shall be produced underaseptic conditions.

In one embodiment a sterile column is produced by packing columns underaseptic conditions by using pre-sterilized components such as columnhousing, medium, process fluids, fluid lines, vessels, containers, bags,sensors. Methods of pre-sterilisation include autoclaving, steaming (inplace), gamma irradiation, E-Beam irradiation, chemical sterilization(i.e. ethylene oxide) or other methods. The production of columns underaseptic conditions using pre-sterilized components is for examplepreferable if the application of different sterilization methods for thedifferent components of the packed column are preferable or required.For example, the effective pre-sterilisation of chromatography mediummay be done by autoclaving, while autoclaving may be prohibitive as amethod of sterilization for a disposable column housing made of plasticmaterials for reason of cost efficiency and disposability, the latterfor example being the full compatibility with incineration methods forpost use destruction and disposal. In another example, the effectivepre-sterilisation of chromatography medium may require thechromatography medium being sterilized in a certain volume or containershape to uniformly and gently apply the sterilization effect. Thus, thecontainer or bag used for the pre-sterilisation process may be optimizedfor the sterilization and different from the shape and volume of thefinal column otherwise deployed for a sterilization after production ofa packed column.

When producing a sterile column under aseptic conditions, saidcomponents in contact with medium and processing fluid used in theprocess are pre-sterilized, too. For connecting and disconnectingcomponents welding methods may be used. Preferably, sterile connectorsand disconnectors are used for connecting and disconnecting components,such as for example ReadyMate connectors (GE Healthcare).

In one embodiment of the invention an aseptic packed column is achievedby packing a pre-sterilized axial flow chromatography column with a bedof pre-sterilized particulate medium of a target bed height. Said columncomprises a housing comprising an elongated tubular side wall andopposed, axially spaced first and second end units separated by saidside wall. At least one said unit is axially movable by drive meansrelative to the other said unit. Said drive units could be non-integralto and external to the column or alternatively said drive means could beintegral to the column. Said column further comprises a first filteradjacent to said first unit and a second filter adjacent to said secondunit which together with the side wall define an enclosed bed space forcontaining a bed of particulate medium and wherein relative movement ofthe first and/or second filter alters the bed height. Said first endunit comprises a first valve means for filling of the bed space withsaid particulate medium and a first port for adding a liquid to orremoving a liquid from the bed space. The second end unit comprises asecond port for adding a liquid to or removing a liquid from the bedspace.

The packing method comprises:

-   -   i. adjusting the axial spacing between the first and second        filters to a distance greater than that of the target bed        height;    -   ii. introducing a suspension of the pre-sterilized particulate        medium into the bed space via the first valve means to provide a        bed of particulate medium therein;    -   iii. compressing said bed of particulate medium to produce the        target bed height by axial movement of the first and/or second        end filters.

This described packing method where pre-sterilized particulate medium isprovided into a pre-sterilized column will provide an aseptic packedcolumn. Suitable aseptic, disposable components are used, like bags,tubing and connectors.

Either external drive means together with tie rods can be used asdescribed above. However in this embodiment a drive means that isintegrated into the column could alternatively be used. Such drive meanscould be for example in the form of an hydraulic chamber in the columnadjacent to a movable adapter. Here, the adapter may be fixedmechanically (locking ring, tie bars etc.) after the hydraulic actionand adjuster adjustment so that the chamber may be drained from liquid.Another alternative could be to use threaded tie bars that allow amechanical adjustment of the adapter height by adjustment of the nuts onthe tie bar. Here, the tie bar could be built from segments such that anextending segment used for the adapter movement is removed after theadapter adjustment. Another alternative would be to use spring loadedadapter movement, whereby the final adapter position may be pre-definedby a mechanical stop hereby limiting the stroke due to the springaction. The spring action may be mechanically released to initiate theadapter movement. By throtteling the fluid flow of the excess fluidremoved from the column space during the adapter movement, the speed ofmovement can be controlled. Further the final position of the adaptercan be controlled temporarily by blocking the flow of said excess liquidsuch that a mechanical locking mechanism can be brought in place. Otheralternatives of driving means could be hydraulic cylinders or electricaldrive motor.

The principle of pre-sterilizing the chromatography column before analready pre-sterilized chromatography media is introduced to the columnvia aseptic equipment can be adopted also for other types ofchromatography columns than has been described above. For example thiswould be suitable also for chromatography columns without driving meanswhere the packing is provided by introducing chromatography media slurrythrough the nozzle into the column having a fixed volume. If an adaptoris provided the adapter has been provided at the wanted bed heightbeforehand. The slurry is provided into the column until the bed ispacked.

The present invention is also related to a method for providing anaseptic chromatography column where said method comprises the steps of:

-   -   pre-sterilize an empty chromatography column;    -   pre-sterilize a chromatography medium;    -   introducing the pre-sterilized chromatography medium into the        pre-sterilized chromatography column using aseptic equipment,        thereby providing an aseptic chromatography column comprising        chromatography medium.        With this method it is possible to use different types or        different intensities of sterilization for the sterilization of        the column and the media.

The method for pre-sterilization of the chromatography column and themethod for pre-sterilisation of the chromatography medium can beselected from at least one of the different methods of sterilizationsuch as heat sterilization by autoclaving or steaming (in place),chemical sterilization (i.e. ethylene oxide), gamma irradiation or eBeamirradiation.

In one embodiment of the invention the method for sterilization used forthe pre-sterilization of the chromatography column is different from themethod for sterilization used for the pre-sterilization of thechromatography medium. This can be advantageous when one method forsterilization is not suitable for sterilizing both the column and themedia. For example gamma radiation may be a suitable method forsterilizing the chromatography column but not for sterilizing the mediaor autoclaving may be suitable method for sterilization for the mediabut not for the column, especially when plastic materials are used asmethod of construction.

In one embodiment of the invention the method for pre-sterilization ofthe chromatography column and the method for pre-sterilisation of thechromatography medium are the same whereby the intensity of protocol forperforming the pre-sterilisation using said method are different.

The invention is also related to an aseptic chromatography columncomprising chromatography medium wherein the chromatography column hasbeen pre-sterilized separately and the chromatography medium has beenpre-sterilized separately before introduction into the chromatographycolumn.

In one embodiment of the invention the pre-sterilization ofchromatography medium is performed on dry chromatography medium. Thepre-sterilized dry chromatography medium can be re-suspended beforepacking the pre-sterilized column. In one embodiment, the drychromatography medium is introduced in its dry state into the column andwetted inside the column space after introduction into the column.

FIG. 6 shows the chromatographic separation efficiency by example of atracer pulse experiment achieved on a 10 liter column in accordance withthe invention, operated in both downflow (solid line) and upflow (dottedline) mode. The column was packed with a bed of Capto™ Q anion exchangeresin (GE Healthcare, Uppsala, Sweden) of 85 μm agarose particlediameter using the method according to the invention. The column had avolume of 10.8 L, a diameter of 263 mm and a bed height of 200 mm.Acetone (1% of packed bed volume) was used as a tracer substance andeluted from the column using water as mobile phase and the absorbancemonitored at 280 nm. As can be seen from Table 1 below, excellent columnefficiency was observed with the 85 μm agarose medium used, either indownflow (solid line) or upflow (dotted line) mode.

TABLE 1 Observed Acceptance Plates/meter 4430 >3700 (for 85 μm) (N/m)Reduced plate height 2.5 <3.0 (h) Peak asymmetry 1.14 0.8-1.8 (Af)

The data from Table 1 were derived from the chromatogram of FIG. 6 asdescribed below.

As a measure for column efficiency, the reduced plate height isdetermined with help of the peak width w_(h) at half the height of theeluted peak, as shown in FIG. 7. This procedure is an approximationvalid for a peak with Gaussian shape. In practice, eluted peaks oftendeviate from this ideal gaussian shape and peak skewness is describedqualitatively by a so-called asymmetry factor A_(f), where ‘leading’ inthe RTD is indicated by A_(f)<1 and ‘tailing’ by A_(f)>1. Commonlyapplied acceptance criteria for the asymmetry factor are0.8<A_(f)<1.5-1.8, depending on the type of application.

$\begin{matrix}{{h = {\frac{HETP}{d_{P}} = {\frac{L}{d_{p}}\frac{1}{5.54}\left( \frac{w_{h}}{V_{R}} \right)^{2}}}}{A_{f} = {b\text{/}a}}} & \left( {{see}\mspace{14mu}{{FIG}.\mspace{11mu} 7}} \right)\end{matrix}$A_(f) asymmetry factord_(p) particle diameterh reduced plate heightHETP height equivalent of a theoretical plateL bed height, packed bedu_(s) superficial velocity in packed bedV_(R) retention volumew_(h) peak width at 50% of max. peak height

The invention claimed is:
 1. A method for providing an asepticchromatography column, said method comprising: providing an emptychromatography column having a housing including sidewalls and includinga first end unit forming an external surface of the chromatographycolumn and extending above the sidewalls and including a valve foradmitting chromatography medium in a slurry or suspension and a filterextending across an area of a bed space of the chromatography columnalong an inner surface of the first end unit; pre-sterilizing thechromatography column; providing chromatography medium through the valveinto a bed space within the chromatography column; pre-sterilizing thechromatography medium; adjusting the height of the bed space within thechromatography column by controllably adjusting the first end unit andthe filter thereon by an external drive having a platform attached tothe first end unit, to a specific position at a distance greater thanthat of a target bed height wherein the first end unit is spaced apredetermined distance above the sidewalls of the housing; introducingthe chromatography medium as a suspension or slurry into thechromatography column using aseptic equipment; and compressing a bed ofthe chromatography medium within the chromatography column once the flowof suspension or slurry has stopped by performing axial movement of thefirst end unit and the filter thereon in a downward direction until thefirst end unit rests on the sidewalls of the housing.
 2. The methodaccording to claim 1, wherein the step for pre-sterilizing of thechromatography column and the step of pre-sterilizing of thechromatography medium include at least one of the sterilization methodsof: heat sterilization by autoclaving or steaming (in place); chemicalsterilization; gamma irradiation; and eBeam irradiation.
 3. The methodaccording to claim 2, wherein the sterilization method used for thepre-sterilizing of the chromatography column is different from thesterilization method used for the pre-sterilizing of the chromatographymedium.
 4. The method according to claim 2, wherein the sterilizationmethod of the chromatography column and the sterilization method of thechromatography medium are the same whereby the intensity protocol forperforming those sterilization methods are different.